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Lytic cell death in metabolic liver disease

Published:April 13, 2020DOI:https://doi.org/10.1016/j.jhep.2020.04.001

      Summary

      Regulated cell death is intrinsically associated with inflammatory liver disease and is pivotal in governing outcomes of metabolic liver disease. Different types of cell death may coexist as metabolic liver disease progresses to inflammation, fibrosis, and ultimately cirrhosis. In addition to apoptosis, lytic forms of hepatocellular death, such as necroptosis, pyroptosis and ferroptosis elicit strong inflammatory responses due to cell membrane permeabilisation and release of cellular components, contributing to the recruitment of immune cells and activation of hepatic stellate cells. The control of liver cell death is of fundamental importance and presents novel opportunities for potential therapeutic intervention. This review summarises the underlying mechanism of distinct lytic cell death modes and their commonalities, discusses their relevance to metabolic liver diseases of different aetiologies, and acknowledges the limitations of current knowledge in the field. We focus on the role of hepatocyte necroptosis, pyroptosis and ferroptosis in non-alcoholic fatty liver disease, alcohol-associated liver disease and other metabolic liver disorders, as well as potential therapeutic implications.

      Keywords

      Introduction

      Cells can be exposed to unrecoverable extra- or intracellular perturbations that disrupt cellular homeostasis and activate signal transduction cascades, ultimately leading to cell death and liver injury. The regulated cell death (RCD) modalities are initiated and propagated by specific molecular mechanisms, with considerable interactivity. Moreover, each type of RCD is characterised by distinct morphological, biochemical, and molecular features with specific physiological consequences ranging from anti-inflammatory and tolerogenic to proinflammatory and immunogenic profiles.
      Decades of research have revealed multiple forms of genetically encoded RCD pathways with increasingly recognised relevance in disease. Importantly, these novel RCD pathways can coexist simultaneously in pathological contexts, and several share overlapping mechanisms that can act as a “backup” dying strategy to ensure organism homeostasis when a death-inducing cellular threshold is reached. In chronic liver diseases, viral, toxic, metabolic or autoimmune triggers cause hepatocellular death, followed by inflammation and compensatory proliferation, often closely linked to the development of fibrosis, cirrhosis, and hepatocellular carcinoma.
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      Cell death and cell death responses in liver disease: mechanisms and clinical relevance.
      Traditionally, apoptosis was considered a highly regulated process, diametrically opposed to passive necrosis, itself considered an accidental and uncontrolled form of cell death. Passive necrosis occurs when cells are irreparably damaged by external forces, resulting in rapid cytoplasmic and organelle swelling (oncosis), along with plasma membrane permeabilisation and subsequent leakage of damage-associated molecular patterns (DAMPs) that trigger an immune response. However, recent findings highlight multiple forms of regulated necrotic modalities, including necroptosis, pyroptosis and ferroptosis, that share key morphologic features with passive necrosis yet have well-defined and regulated causal mechanisms.
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      Regulated necrosis: the expanding network of non-apoptotic cell death pathways.
      Necroptosis is probably the best-understood form of regulated necrosis, since it shares multiple molecular components with the extrinsic apoptotic pathway and can act as a fail-safe mechanism to ensure cell death progression when apoptosis is abnormally inhibited, which may also be relevant in pathological conditions in the liver.
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      Nevertheless, other alternative forms of RCD, such as ferroptosis or pyroptosis may turn out to be equally important. Importantly, a better characterisation of RCD in liver disease may lead to novel therapeutic opportunities. As the field continues to progress and novel signalling pathways that orchestrate RCD are still being characterised, this review summarises current knowledge on the contribution of hepatocellular RCD modalities in metabolic liver disease. Experimental models and patient findings are addressed, as well as the pathophysiological relevance of each of the main types of RCD in hepatocytes, controversies and unsolved issues, and potential therapeutic applications. RCD in non-hepatocyte liver cells, immune cells and other cell types is beyond the scope of this review, although it may prove equally important in liver disease.

      The principles of alternative cell death modalities

      Necroptosis

      The first evidence of necroptosis was provided in 1996 by Ray et al., who observed a lytic mode of cell death in pig kidney cells infected with cow pox virus, which was governed by the expression of the viral cytokine response modifier A, a caspase inhibitor.
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      The mode of death of pig kidney cells infected with cowpox virus is governed by the expression of the crmA gene.
      In 2000, Holler et al. demonstrated that FAS, tumour necrosis factor (TNF) receptor 1 (TNFR1) and TNF-related apoptosis-inducing ligand receptor (TRAIL-R), the classical death receptors (DRs), initiated cell death by 2 alternative pathways, one relying on caspase-8 (i.e., the classical extrinsic apoptotic pathway) and the other dependent on the receptor interacting protein kinase 1 (RIPK1) (i.e., necroptosis).
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      Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.
      However, the term used to describe this novel mode of cell death only appeared in 2005, when Degterev et al. showed that necrostatin-1, a chemical compound that blocks the kinase activity of RIPK1, was able to inhibit cell death in TNF-treated cell lines.
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      Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury.
      The 2 downstream core components of the necroptotic machinery, namely RIPK3 and mixed lineage kinase domain like pseudokinase (MLKL), were then identified in less than a decade.
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      Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.
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      Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.
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      RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis.
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      Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis.
      Morphologically, necroptosis, which is primarily triggered by microbial infections and physicochemical stressors (e.g., radiation or chemotherapy), exhibits the features of passive necrosis (e.g., in response to extreme external factors) with increased cell volume, swelling of organelles, loss of membrane integrity and cellular collapse, leading to the release of DAMPs (e.g. high mobility group box 1 [HMGB1]; interleukin [IL]-1α, IL-33). Although no specific necroptotic DAMPs have been identified so far, their release during necroptosis provokes a strong inflammatory response that has been linked to the development of many diseases.
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      Necroptosis in development, inflammation and disease.
      Necroptosis typically occurs upon DR activation (e.g., TNFR1, CD95, TRAIL-R), and only if apoptotic signalling components are inactive, absent or inhibited (e.g., caspase inhibition) (Fig. 1). In this specific context, DR triggers the formation of a RIPK1/RIPK3 cell death platform, named the necrosome.
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      The molecular machinery of regulated cell death.
      Inside, RIPK1 and RIPK3 adopt a hetero-amyloid structure through critical RHIM (RIP homotypic interaction motif) interactions.
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      The structure of the necrosome RIPK1-RIPK3 Core, a human hetero-amyloid signaling complex.
      Consequently, RIPK3 mediates the phosphorylation of MLKL, resulting in MLKL oligomerisation and translocation to the plasma membrane.
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      • Kroemer G.
      The molecular machinery of regulated cell death.
      Recent results suggest that casein kinase 1 family proteins are necrosome components, which are required to directly phosphorylate serine 227 of human RIPK3 and induce necroptosis.
      • Hanna-Addams S.
      • Liu S.
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      • Chen S.
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      CK1alpha, CK1delta, and CK1epsilon are necrosome components which phosphorylate serine 227 of human RIPK3 to activate necroptosis.
      Finally, MLKL forms a pore in the plasma membrane,
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      MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates.
      ,
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      • Tanzer M.C.
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      Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.
      and increases permeability via the activation of ADAM (A disintegrin and metalloproteinase) proteases,
      • Cai Z.
      • Zhang A.
      • Choksi S.
      • Li W.
      • Li T.
      • Zhang X.M.
      • et al.
      Activation of cell-surface proteases promotes necroptosis, inflammation and cell migration.
      Ca2+ influx by targeting the cation channel TRPM7 (transient receptor potential melastatin related 7),
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      • Zhao J.
      • Chiang H.C.
      • Choksi S.
      • Liu J.
      • et al.
      Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis.
      and phosphatidylserine externalisation.
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      • Erlich Z.
      • Hourizadeh A.
      • Ofir-Birin Y.
      • Croker B.A.
      • et al.
      Phosphatidylserine externalization, “necroptotic bodies” release, and phagocytosis during necroptosis.
      Necroptosis activation requires MLKL phosphorylation, which is dependent on the kinase activity of RIPK3, itself dependent on phosphorylation by RIPK1; hence, RIPK1 and/or 3 phosphorylation cannot be equated with necroptosis without MLKL phosphorylation. There is considerable crosstalk between apoptosis and necroptosis in DR signalling pathways,
      • Vanden Berghe T.
      • Kaiser W.J.
      • Bertrand M.J.
      • Vandenabeele P.
      Molecular crosstalk between apoptosis, necroptosis, and survival signaling.
      and it seems that one cannot be achieved without inhibiting the other. On one hand, caspase-8 cleaves and inactivates RIPK1 and RIPK3, which suppresses necroptosis.
      • Lin Y.
      • Devin A.
      • Rodriguez Y.
      • Liu Z.G.
      Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis.
      ,
      • Feng S.
      • Yang Y.
      • Mei Y.
      • Ma L.
      • Zhu D.E.
      • Hoti N.
      • et al.
      Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain.
      On the other hand, the activity of RIPK3 determines whether cells die by necroptosis (i.e., the necrosome formed) or apoptosis (i.e., kinase activity of RIPK3 inhibited).
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      • Kapoor N.
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      Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis.
      ,
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      • Huang C.
      • Guo H.
      • et al.
      RIP3 induces apoptosis independent of pronecrotic kinase activity.
      Figure thumbnail gr1
      Fig. 1RIPK3- and MLKL-dependent necroptosis mediated by various stimuli.
      Binding of TNF-α or FasL to their receptor induces the formation of a receptor proximal complex (complex 1), which consists of adaptor proteins (e.g., TRADD, FADD, TAB), ubiquitin ligases (e.g., TRAF2, LUBAC, cIAPs), and kinases (e.g., TAK and RIPK1). The ubiquitination of RIPK1 provides docking sites for the recruitment of key proteins leading to cell survival by NF-κB-dependent upregulation of pro-survival genes. Then, complex 1 internalisation leads to the formation of complex 2a, which ultimately results in caspase-8-dependent apoptosis. Finally, when caspases or cIAPs are inhibited, RIPK1 associates with RIPK3 to form the necrosome (complex 2b), which in turn recruits the pseudokinase MLKL. RIPK3-mediated phosphorylation of MLKL results in MLKL translocation to the plasma membrane and pore formation. Other necroptotic stimuli such as IFNα/β, virus, pathogen-associated molecular patterns (e.g., LPS, poly I:C) are also depicted. Although the nature of these signalling complexes is not completely characterised, the main mediators activating the necrosome are shown. To antagonise necroptosis, ESCRT-III components are recruited to localised sites of MLKL-directed membrane damage to shed broken membrane into blebs. cIAPs, cellular inhibitors of apoptosis; dsRNA, double-stranded RNA; ESCRT, endosomal sorting complex required for transport; FADD, FAS-associated protein with death domain; HMGB1, high mobility group box 1; IFN, interferon; IFNR, interferon-receptor; IL-, interleukin-; LPS, lipopolysaccharide; LUBAC, linear ubiquitin chain assembly complex; MLKL, mixed lineage kinase domain like; PKR, protein kinase R; RIPK, receptor interacting protein kinase; TAB, transforming growth factor beta-binding protein; TAK, transforming growth factor beta-activated kinase 1; TLR, toll-like receptor; TNF, tumour necrosis factor; TNFR1, TNF receptor 1; TRADD, TNF receptor type 1-associated death domain; TRAIL, TNF-related apoptosis-inducing ligand receptor; TRAF2, TNF receptor-associated factor 2; ZBP1, Z-DNA binding protein 1.
      The activation of necroptosis is not limited to DRs, and it can be triggered by multiple pathways, including toll-like receptors (e.g., TLR3 and TLR4),
      • He S.
      • Liang Y.
      • Shao F.
      • Wang X.
      Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway.
      nucleic acid sensors (e.g., Z-DNA-binding protein 1 [ZBP1, also known as DAI]),
      • Upton J.W.
      • Kaiser W.J.
      • Mocarski E.S.
      DAI/ZBP1/DLM-1 complexes with RIP3 to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vIRA.
      retinoic acid inducible gene 1 protein (RIG1, also known as DDX58),
      • Schock S.N.
      • Chandra N.V.
      • Sun Y.
      • Irie T.
      • Kitagawa Y.
      • Gotoh B.
      • et al.
      Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway.
      transmembrane protein 173 (TMEM173, also known as STING),
      • Brault M.
      • Olsen T.M.
      • Martinez J.
      • Stetson D.B.
      • Oberst A.
      Intracellular nucleic acid sensing triggers necroptosis through synergistic type I IFN and TNF signaling.
      and adhesion receptors.
      • Wang X.
      • He Z.
      • Liu H.
      • Yousefi S.
      • Simon H.U.
      Neutrophil necroptosis is triggered by ligation of adhesion molecules following GM-CSF priming.
      These signalling pathways are often independent of RIPK1, but all involve the phosphorylation of MLKL by RIPK3 and MLKL-mediated pore formation in the plasma membrane.
      • Tang D.
      • Kang R.
      • Berghe T.V.
      • Vandenabeele P.
      • Kroemer G.
      The molecular machinery of regulated cell death.
      Interestingly, the endosomal sorting complexes required for transport (ESCRT)-III complex, a membrane remodelling and scission machinery, limit MLKL-mediated necroptosis by promoting membrane repair.
      • Gong Y.N.
      • Guy C.
      • Olauson H.
      • Becker J.U.
      • Yang M.
      • Fitzgerald P.
      • et al.
      ESCRT-III Acts downstream of MLKL to regulate necroptotic cell death and its consequences.
      As MLKL also regulates endosomal trafficking and extracellular vesicle generation,
      • Yoon S.
      • Kovalenko A.
      • Bogdanov K.
      • Wallach D.
      MLKL, the protein that mediates necroptosis, also regulates endosomal trafficking and extracellular vesicle generation.
      a delicate balance between membrane injury and repair exists and ultimately decides cell fate in necroptosis.
      Regulated hepatocyte cell death drives metabolic liver disease progression to liver inflammation, fibrosis and cirrhosis.

      Pyroptosis

      Pyroptosis was initially observed in 1992 by Zychlinsky et al., who described a lytic form of cell death in Shigella flexneri-infected macrophages.
      • Zychlinsky A.
      • Prevost M.C.
      • Sansonetti P.J.
      Shigella flexneri induces apoptosis in infected macrophages.
      However, the term ‘pyroptosis’ only emerged nearly a decade later, in 2001, when Cookson and Brennan demonstrated that Salmonella-induced macrophage death was dependent on caspase-1.
      • Cookson B.T.
      • Brennan M.A.
      Pro-inflammatory programmed cell death.
      Since this discovery, the number of pyroptotic caspases (as opposed to apoptotic caspases) has considerably increased, including caspase-1, caspase-11 and its human orthologs caspase-4 and 5,
      • Kayagaki N.
      • Warming S.
      • Lamkanfi M.
      • Vande Walle L.
      • Louie S.
      • Dong J.
      • et al.
      Non-canonical inflammasome activation targets caspase-11.
      ,
      • Shi J.
      • Zhao Y.
      • Wang Y.
      • Gao W.
      • Ding J.
      • Li P.
      • et al.
      Inflammatory caspases are innate immune receptors for intracellular LPS.
      and even more surprisingly the apoptotic effector caspase-3 under specific circumstances.
      • Rogers C.
      • Fernandes-Alnemri T.
      • Mayes L.
      • Alnemri D.
      • Cingolani G.
      • Alnemri E.S.
      Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death.
      ,
      • Wang Y.
      • Gao W.
      • Shi X.
      • Ding J.
      • Liu W.
      • He H.
      • et al.
      Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin.
      Pyroptosis, which occurs primarily upon infection with intracellular pathogens, is characterised by caspase-dependent pore formation in the plasma membrane, swelling, rupture of the cell, and release of proinflammatory IL-1β and IL-18.
      • Bergsbaken T.
      • Fink S.L.
      • Cookson B.T.
      Pyroptosis: host cell death and inflammation.
      In 2015, the pore-forming gasdermin D (GSDMD) was identified as the executioner of pyroptosis.
      • Kayagaki N.
      • Stowe I.B.
      • Lee B.L.
      • O'Rourke K.
      • Anderson K.
      • Warming S.
      • et al.
      Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.
      ,
      • Shi J.
      • Zhao Y.
      • Wang K.
      • Shi X.
      • Wang Y.
      • Huang H.
      • et al.
      Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.
      Caspases-1 and -11 cleave GSDMD into a 31-kDa N-terminal GSDMDNT fragment, which exhibits intrinsic pore-forming activity, and a 22-kDa C-terminal GSDMDCT fragment that binds to GSDMDNT to maintain the protein in an inhibitory state.
      • Kayagaki N.
      • Stowe I.B.
      • Lee B.L.
      • O'Rourke K.
      • Anderson K.
      • Warming S.
      • et al.
      Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.
      ,
      • Shi J.
      • Zhao Y.
      • Wang K.
      • Shi X.
      • Wang Y.
      • Huang H.
      • et al.
      Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.
      The overexpression of GSDMDNT alone results in induction of pyroptosis, whereas that of GSDMDCT blocks GSDMDNT-induced pyroptosis.
      • Shi J.
      • Zhao Y.
      • Wang K.
      • Shi X.
      • Wang Y.
      • Huang H.
      • et al.
      Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.
      Interestingly, GSDMD belongs to a larger protein family, which consists of GSDMA, GSDMB, GSDMC, GSDMD, GSDME (also referred to as DFNA5), and DFNB59.
      • Orning P.
      • Lien E.
      • Fitzgerald K.A.
      Gasdermins and their role in immunity and inflammation.
      Recently, GSDME was identified as an additional executioner of pyroptosis, able to switch caspase-3-mediated apoptosis (induced by TNF or chemotherapy drugs) to pyroptosis.
      • Wang Y.
      • Gao W.
      • Shi X.
      • Ding J.
      • Liu W.
      • He H.
      • et al.
      Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin.
      Although most of the gasdermins have been associated with the occurrence and development of various diseases, their precise function and molecular mechanism of activation remain largely unknown.
      • Broz P.
      • Pelegrin P.
      • Shao F.
      The gasdermins, a protein family executing cell death and inflammation.
      Pyroptosis is activated by canonical and non-canonical signalling pathways, which differ in the use of cytoplasmic multiprotein complexes named inflammasomes (Fig. 2).
      • Broz P.
      • Dixit V.M.
      Inflammasomes: mechanism of assembly, regulation and signalling.
      ,
      • Lamkanfi M.
      • Dixit V.M.
      Mechanisms and functions of inflammasomes.
      The canonical pathway begins with inflammasomes that recognise various exogenous and endogenous danger signals, including DAMPs and pathogen-associated molecular patterns (PAMPs). The canonical inflammasomes comprise a sensor protein belonging to the nucleotide-binding domain and leucine-rich-repeat (LRR)-containing (NLR) family, or the AIM2-like receptor or pyrin family; an adaptor protein, ASC (also called PYCARD); and an inactive zymogen, pro-caspase-1.
      • Vanaja S.K.
      • Rathinam V.A.
      • Fitzgerald K.A.
      Mechanisms of inflammasome activation: recent advances and novel insights.
      When formed, canonical inflammasomes lead to the activation of caspase-1 that cleaves pro-IL-1β and pro-IL-18 into their active forms. Then, IL-1β and IL-18 are released extracellularly as a result of pore formation in the plasma membrane by GSDMDNT. On the other side, the non-canonical pathway is dependent on caspase-11, which can cleave GSDMD independently of inflammasome priming. In the latter case, GSDMDNT signals back to the canonical NLRP3 inflammasome, which in turn activates the caspase-1-dependent pathway.
      • Broz P.
      • Dixit V.M.
      Inflammasomes: mechanism of assembly, regulation and signalling.
      • Lamkanfi M.
      • Dixit V.M.
      Mechanisms and functions of inflammasomes.
      • Vanaja S.K.
      • Rathinam V.A.
      • Fitzgerald K.A.
      Mechanisms of inflammasome activation: recent advances and novel insights.
      Figure thumbnail gr2
      Fig. 2Caspase-1-dependent and -independent pyroptosis.
      Caspase-1-dependent pyroptosis requires activation of the canonical inflammasomes, including NLRP1b, NLRP3, NLRC4, AIM2 and Pyrin. The NLRs are characterised by the combined presence of a nucleotide-binding and oligomerization domain (NACHT) and a variable number of LRRs. They also contain either a CARD or PYD in their amino terminus. The AIM2 protein is composed of a N-terminal PYD and a C-terminal DNA-binding HIN200 domain. The pyrin protein has a N-terminal PYD, a bZIP transcription factor domain, a B-box, a coiled-coil, and a C-terminal B30.2 domain. NLRP1b and NLRC4 recruit caspase-1 via their CARD domain, and the bipartite PYD-CARD adaptor protein ASC is required for assembly of the AIM2, NLRP3 and pyrin inflammasomes. The pyrin protein also binds directly to caspase-1 via its B30.2 domain. Caspase-1-independent pyroptosis requires the activation of caspase-11, which recognises cytosolic lipopolysaccharide and cleaves GSDMD to initiate pyroptosis. In this pathway, GSDMDNT also activates the NLRP3 inflammasome and thereby caspase-1-dependent maturation of IL-1β and IL-18. Caspase-3-dependent pyroptosis requires the activation of GSDME. Caspase-3 can be activated by the mitochondrial and death receptor pathway. Active caspase-3 cleaves GSDME, to produce GSDMENT, which then forms pores in the plasma membrane. AIM2, absent in melanoma 2; CARD, caspase recruitment domain; dsRNA, double-stranded RNA; GSDMD/E, gasdermin D/E; IL-, interleukin; LRR, leucine-rich-repeat; PYD, pyrin domain.

      Ferroptosis

      Ferroptosis was originally observed in 2003 using erastin, a cell-permeable compound, which was only lethal to engineered human tumour cells expressing an oncogenic RAS mutation.
      • Dolma S.
      • Lessnick S.L.
      • Hahn W.C.
      • Stockwell B.R.
      Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells.
      The term ferroptosis was coined in 2012 by the Stockwell lab to describe non-apoptotic cell death caused by the accumulation of iron-dependent lipid peroxides, induced by erastin.
      • Dixon S.J.
      • Lemberg K.M.
      • Lamprecht M.R.
      • Skouta R.
      • Zaitsev E.M.
      • Gleason C.E.
      • et al.
      Ferroptosis: an iron-dependent form of nonapoptotic cell death.
      Ferroptosis, which principally exerts tumour-suppressor functions, is morphologically characterised by cell volume shrinkage and increased mitochondrial membrane density without typical apoptotic or necrotic manifestations.
      • Dixon S.J.
      • Lemberg K.M.
      • Lamprecht M.R.
      • Skouta R.
      • Zaitsev E.M.
      • Gleason C.E.
      • et al.
      Ferroptosis: an iron-dependent form of nonapoptotic cell death.
      Ferroptosis can be induced either in a canonical way by a direct or indirect inactivation of the glutathione peroxidase 4 (GPX4), the major protective mechanism of biological membranes against peroxidation damage, or in a non-canonical manner by increasing the labile iron pool.
      • Dixon S.J.
      • Lemberg K.M.
      • Lamprecht M.R.
      • Skouta R.
      • Zaitsev E.M.
      • Gleason C.E.
      • et al.
      Ferroptosis: an iron-dependent form of nonapoptotic cell death.
      The pathological relevance of ferroptosis has been evaluated using ferrostatin-1, a lipid reactive oxygen species (ROS) scavenger that prevents ferroptosis induced by erastin.
      • Dixon S.J.
      • Lemberg K.M.
      • Lamprecht M.R.
      • Skouta R.
      • Zaitsev E.M.
      • Gleason C.E.
      • et al.
      Ferroptosis: an iron-dependent form of nonapoptotic cell death.
      • Skouta R.
      • Dixon S.J.
      • Wang J.
      • Dunn D.E.
      • Orman M.
      • Shimada K.
      • et al.
      Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models.
      • Xie Y.
      • Hou W.
      • Song X.
      • Yu Y.
      • Huang J.
      • Sun X.
      • et al.
      Ferroptosis: process and function.
      In the canonical pathway, erastin interacts directly with the transporter solute carrier family 7 member 5 (SLC7A5), which disrupts the transport of amino acids into the cell by system Xc- (Fig. 3).
      • Dixon S.J.
      • Lemberg K.M.
      • Lamprecht M.R.
      • Skouta R.
      • Zaitsev E.M.
      • Gleason C.E.
      • et al.
      Ferroptosis: an iron-dependent form of nonapoptotic cell death.
      The Xc- transporter system is composed of a regulatory subunit solute carrier family 3 member 2 (SLC3A2) and a catalytic subunit solute carrier family 7 member 11 (SLC7A11), which promotes the cellular uptake of cystine, the plasma precursor of cysteine, by exchange with glutamate.
      • Lo M.
      • Wang Y.Z.
      • Gout P.W.
      The x(c)- cystine/glutamate antiporter: a potential target for therapy of cancer and other diseases.
      In cells, cysteine is required for the synthesis of glutathione (GSH), a major redox regulatory system.
      • Lu S.C.
      Glutathione synthesis.
      By indirectly blocking the Xc- transporter, erastin inhibits GSH synthesis, which is used by GPX4 to eliminate the production of phospholipid hydroperoxides (PL-OOH), the major mediator of chain reactions in lipoxygenases (LOXs).
      • Yang W.S.
      • SriRamaratnam R.
      • Welsch M.E.
      • Shimada K.
      • Skouta R.
      • Viswanathan V.S.
      • et al.
      Regulation of ferroptotic cancer cell death by GPX4.
      System Xc- inhibitors (e.g., erastin, sulfasalazine, sorafenib and L-glutamate) are considered as class I ferroptosis inducers, whereas direct GPX4 inhibitors (e.g., RAS-selective lethality protein 3, ML162 and altretamine) are referred to as class II inducers.
      • Xie Y.
      • Hou W.
      • Song X.
      • Yu Y.
      • Huang J.
      • Sun X.
      • et al.
      Ferroptosis: process and function.
      Figure thumbnail gr3
      Fig. 3The core components controlling ferroptosis.
      GSH is a tripeptide essential for preventing damage caused by ROS, such as lipid peroxides. It is synthesised continuously from cysteine, glutamate and glycine. Cellular availability of cysteine is the limiting step for GSH synthesis. The Xc- transporter, which consists of 2 subunits belonging to the family of solute transporters (SLC3A2 and SLC7A11), is responsible for the uptake of extracellular cystine, the precursor of cysteine. GSH provides electrons to the key regulator of ferroptosis, GPX4, which reduces lipid peroxides (-OOH) in plasma membranes to the corresponding alcohols (-OH). Recently, the enzymes ACSL4 and LPCAT3 that are directly involved in shaping the cellular lipid composition, have been shown to sensitise cells to ferroptosis. Oxidation of lipid bilayers during ferroptosis occurs both in an enzymatic (i.e., LOXs) and non-enzymatic (i.e., radical-mediated autoxidative; symbolized by the red arrow) manner. Alternatively, FSP1 can suppress ferroptosis by ubiquinone, the reduced form of ubiquinol, which traps lipid peroxyl radicals. FSP1 catalyses the regeneration of ubiquinone using NAD(P)H. Cellular iron homeostasis, which is dependent on the coordination of iron uptake (i.e., TFRC) and export (i.e., ferroportin), directly regulates ferroptosis through Fenton reactions and generation of ROS. Fe3+ is reduced to Fe2+ by metalloreductases in the endosome, and the DMT1 mediates the transport of Fe2+ from the endosome into a labile iron pool in the cytoplasm. RTK activates the oncogene RAS, which is known to induce oxidative stress through generation of ROS. Ferroptosis-inducing drugs are depicted in red, whereas ferroptosis inhibitors are shown in green. ACSL4, acyl-CoA synthetase long chain 4; DMT1, divalent metal transporter 1; FSP1, ferroptosis suppressor protein 1; GPX4, glutathione peroxidase 4; GSH, glutathione; GSSG, glutathione disulfide; LOXs, lipoxygenases; LPCAT3, lysophosphatidylcholine acyltransferase 3; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SLC, solute carriers; TFRC, transferrin receptor.
      The main ROS produced by cellular metabolism or accumulated upon cell transformation (e.g., RAS-mediated) are the superoxide radical anion (O2.-) and hydrogen peroxide (H2O2). In the presence of free iron, these ROS are likely to be converted to hydroxyl radical (HO.), which is highly reactive to macromolecules (e.g., polyunsaturated-fatty-acids [PUFAs]). The reactions involving iron and leading to the hydroxyl or alkoxyl (RO.) radical are traditionally called Fenton reactions.
      • Feng H.
      • Stockwell B.R.
      Unsolved mysteries: how does lipid peroxidation cause ferroptosis?.
      The oxidation of PUFAs, including arachidonic acid, by a catalytic pathway involving acyl-CoA synthetase long-chain family member 4 (ACSL4), lysophosphatidylcholine acyltransferase 3 (LPCAT3), and LOXs, is required for lipotoxicity in ferroptosis.
      • Doll S.
      • Proneth B.
      • Tyurina Y.Y.
      • Panzilius E.
      • Kobayashi S.
      • Ingold I.
      • et al.
      ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.
      • Kagan V.E.
      • Mao G.
      • Qu F.
      • Angeli J.P.
      • Doll S.
      • Croix C.S.
      • et al.
      Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis.
      • Yang W.S.
      • Kim K.J.
      • Gaschler M.M.
      • Patel M.
      • Shchepinov M.S.
      • Stockwell B.R.
      Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis.
      Compared with other proteins of its family, GPX4 is the only member able to reduce membrane phospholipid hydroperoxides, highlighting its key role against lipid peroxidation, which is prominent in ferroptosis and contributes to plasma membrane permeabilisation and consequently release of DAMPs – though the molecular mechanisms remain elusive.
      • Dixon S.J.
      • Lemberg K.M.
      • Lamprecht M.R.
      • Skouta R.
      • Zaitsev E.M.
      • Gleason C.E.
      • et al.
      Ferroptosis: an iron-dependent form of nonapoptotic cell death.
      Recently, the ferroptosis suppressor protein 1 (FSP1, previously known as apoptosis-inducing factor mitochondrial 2) was identified as a key component of a non-mitochondrial CoQ10 (ubiquinone) antioxidant system that acts in parallel to the GPX4 canonical pathway to halt the progression of lipid peroxides.
      • Doll S.
      • Freitas F.P.
      • Shah R.
      • Aldrovandi M.
      • da Silva M.C.
      • Ingold I.
      • et al.
      FSP1 is a glutathione-independent ferroptosis suppressor.
      ,
      • Bersuker K.
      • Hendricks J.M.
      • Li Z.
      • Magtanong L.
      • Ford B.
      • Tang P.H.
      • et al.
      The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.
      In the non-canonical pathway, increased iron uptake by the transferrin receptor and reduced iron export by ferroportin promote oxidative damage and ferroptosis.
      • Gao M.
      • Monian P.
      • Quadri N.
      • Ramasamy R.
      • Jiang X.
      Glutaminolysis and transferrin regulate ferroptosis.
      ,
      • Geng N.
      • Shi B.J.
      • Li S.L.
      • Zhong Z.Y.
      • Li Y.C.
      • Xua W.L.
      • et al.
      Knockdown of ferroportin accelerates erastin-induced ferroptosis in neuroblastoma cells.

      Commonalities

      Unlike necrosis, which is accidental and uncontrolled, lytic RCD, including necroptosis, pyroptosis and ferroptosis, are genetically defined and employ complex machinery specifically designed to perforate the plasma membrane. This common phenomenon plays an evolutionarily conserved role in systemic immunity, combining the killing of cells with alerting the immune system through the release of DAMPs. Although these 3 forms of lytic RCD are at first glance dedicated to eliminating intracellular pathogens (i.e., pyroptosis and necroptosis) or mutation-driven cancer cells (i.e., ferroptosis), their chronic stimulation can lead to excessive inflammation that may be detrimental and start damaging healthy cells, tissues, and organs.
      • Kolb J.P.
      • Oguin 3rd, T.H.
      • Oberst A.
      • Martinez J.
      Programmed cell death and inflammation: winter is coming.
      In fact, these 3 forms of RCD have already been shown to be involved in the development of many inflammatory diseases from different aetiologies, including liver diseases.
      • Aizawa S.
      • Brar G.
      • Tsukamoto H.
      Cell death and liver disease.
      Accumulating evidence indicates that a chronic low-level of inflammation (also known as sterile-inflammation) is mediated by DAMPs.
      • Chen G.Y.
      • Nunez G.
      Sterile inflammation: sensing and reacting to damage.
      As DAMPs are hallmarks of lytic RCD, and are recognised by pattern recognition receptors, which are also used to induce lytic RCD (e.g., TLRs and NLRs), DAMPs appear to be the common denominators connecting lytic RCD pathways to each other.
      • Chen G.Y.
      • Nunez G.
      Sterile inflammation: sensing and reacting to damage.
      A substantial number of DAMPs, including HMGB1, heat shock proteins, ATP, and interleukins, have been differentially involved in pyroptosis, necroptosis or ferroptosis.
      • Mou Y.
      • Wang J.
      • Wu J.
      • He D.
      • Zhang C.
      • Duan C.
      • et al.
      Ferroptosis, a new form of cell death: opportunities and challenges in cancer.
      ,
      • Frank D.
      • Vince J.E.
      Pyroptosis versus necroptosis: similarities, differences, and crosstalk.
      However, further investigations are needed to identify which specific DAMPs might preferentially activate one lytic RCD or another.
      Recent evidence suggests that caspase-8 may represent the molecular switch that controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage.
      • Fritsch M.
      • Gunther S.D.
      • Schwarzer R.
      • Albert M.C.
      • Schorn F.
      • Werthenbach J.P.
      • et al.
      Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis.
      ,
      • Newton K.
      • Wickliffe K.E.
      • Maltzman A.
      • Dugger D.L.
      • Reja R.
      • Zhang Y.
      • et al.
      Activity of caspase-8 determines plasticity between cell death pathways.
      In addition to its enzymatic role in apoptosis and necroptosis, caspase-8 acts as a scaffolding protein that together with MLKL regulates NLRP3 inflammasome activation in macrophages.
      • Kang S.
      • Fernandes-Alnemri T.
      • Rogers C.
      • Mayes L.
      • Wang Y.
      • Dillon C.
      • et al.
      Caspase-8 scaffolding function and MLKL regulate NLRP3 inflammasome activation downstream of TLR3.
      These unexpected roles of caspase-8 involve the activation of the inflammasome and induction of pyroptosis under circumstances in which apoptosis and necroptosis are compromised, such as when the abundance of viral inhibitors may have driven the counteradaptation of pyroptosis as a host defence.
      • Mocarski E.S.
      • Upton J.W.
      • Kaiser W.J.
      Viral infection and the evolution of caspase 8-regulated apoptotic and necrotic death pathways.
      Questions that remain to be addressed include how the different modes of RCD influence disease progression and coordinate adaptive responses.
      Accumulating evidence suggests that lytic cell death modalities such as hepatocyte necroptosis, pyroptosis and ferroptosis play pathophysiological roles in metabolic liver disease, while other functions in non-hepatocyte liver cells, immune cells and others have been suggested.
      In the next section, we describe the potential roles of RCD in various metabolic liver diseases using an unexpurgated approach, in that the observations described as regulating RCD in these models are included regardless of whether they have been confirmed or whether they are viewed sceptically.

      Alternative cell death modalities in metabolic liver diseases

      Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis

      Non-alcoholic fatty liver disease (NAFLD) is a spectrum of chronic liver disease that ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) and is strongly associated with obesity and the metabolic syndrome. NAFLD is estimated to affect up to one-third of the general population worldwide.
      • Tsochatzis E.A.
      • Newsome P.N.
      Non-alcoholic fatty liver disease and the interface between primary and secondary care.
      Although metabolic alterations caused by free fatty acid overload are considered a major cause of hepatocyte damage, the mechanisms driving disease progression from simple steatosis to NASH remain incompletely understood.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      Indeed, lipotoxicity, oxidative stress, organelle dysfunction and inflammatory responses exacerbate ballooning and increase hepatocyte cell death.
      • Afonso M.B.
      • Castro R.E.
      • Rodrigues C.M.P.
      Processes exacerbating apoptosis in non-alcoholic steatohepatitis.
      Chronic liver cell death may then trigger immune cell recruitment and hepatic stellate cell activation, hepatocyte turnover and clonal expansion, in a feed-forward loop that drives NAFLD progression. While historically much emphasis was placed on apoptosis and necrosis, more recently other types of RCD, such as necroptosis or pyroptosis, controlled by overlapping molecular machineries, and capable of acting as a backup for each other, have been implicated in NAFLD and NASH pathogenesis.

      Necroptosis

      Hepatocellular necroptosis has been described in human NASH and the expression of RIPK3 and MLKL is increased in the livers of patients with biopsy-proven NASH and other chronic liver diseases.
      • Afonso M.B.
      • Rodrigues P.M.
      • Carvalho T.
      • Caridade M.
      • Borralho P.
      • Cortez-Pinto H.
      • et al.
      Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis.
      • Gautheron J.
      • Vucur M.
      • Reisinger F.
      • Cardenas D.V.
      • Roderburg C.
      • Koppe C.
      • et al.
      A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis.
      • Afonso M.B.
      • Rodrigues P.M.
      • Simao A.L.
      • Ofengeim D.
      • Carvalho T.
      • Amaral J.D.
      • et al.
      Activation of necroptosis in human and experimental cholestasis.
      • Gautheron J.
      • Vucur M.
      • Luedde T.
      Necroptosis in nonalcoholic steatohepatitis.
      The systemic release of necrosome proteins RIPK1 and MLKL in patients displaying disease activity further supports the clinical importance of this pathway in NASH.
      • Majdi A.
      • Aoudjehane L.
      • Ratziu V.
      • Islam T.
      • Afonso M.B.
      • Conti F.
      • et al.
      Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease.
      RIPK3 and MLKL expression was also increased in visceral adipose tissue from patients with obesity and type II diabetes, while RIPK3 expression correlated with MLKL and metabolic serum markers.
      • Gautheron J.
      • Vucur M.
      • Schneider A.T.
      • Severi I.
      • Roderburg C.
      • Roy S.
      • et al.
      The necroptosis-inducing kinase RIPK3 dampens adipose tissue inflammation and glucose intolerance.
      High levels of core components of necroptosis were detected in liver, adipose tissue and muscle in diabetic mice, whereas targeting diabetic mice with RIPK1 inhibitors or depletion of MLKL prevented insulin resistance and glucose intolerance, despite no effect on inflammation.
      • Xu H.
      • Du X.
      • Liu G.
      • Huang S.
      • Du W.
      • Zou S.
      • et al.
      The pseudokinase MLKL regulates hepatic insulin sensitivity independently of inflammation.
      Indeed, both specific knockdown of RIPK1, RIPK3 or MLKL and the use of chemical inhibitors activated insulin-stimulated AKT signalling, which is key in glucose homeostasis. Further evidence from animal studies confirmed that RIPK3 increases in the livers of experimental models of NASH.
      • Afonso M.B.
      • Rodrigues P.M.
      • Carvalho T.
      • Caridade M.
      • Borralho P.
      • Cortez-Pinto H.
      • et al.
      Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis.
      ,
      • Gautheron J.
      • Vucur M.
      • Reisinger F.
      • Cardenas D.V.
      • Roderburg C.
      • Koppe C.
      • et al.
      A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis.
      ,
      • Roychowdhury S.
      • McCullough R.L.
      • Sanz-Garcia C.
      • Saikia P.
      • Alkhouri N.
      • Matloob A.
      • et al.
      Receptor interacting protein 3 protects mice from high-fat diet-induced liver injury.
      JNK activation was implicated in RIPK3-dependent liver damage and fibrosis in methionine- and choline-deficient (MCD) diet-fed mice, as inhibition of JNK diminished RIPK3 levels in the liver.
      • Gautheron J.
      • Vucur M.
      • Reisinger F.
      • Cardenas D.V.
      • Roderburg C.
      • Koppe C.
      • et al.
      A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis.
      Similarly, in common bile duct ligation models of hepatic injury, necroptosis was induced and contributed to fibrosis,
      • Afonso M.B.
      • Rodrigues P.M.
      • Simao A.L.
      • Gaspar M.M.
      • Carvalho T.
      • Borralho P.
      • et al.
      miRNA-21 ablation protects against liver injury and necroptosis in cholestasis.
      although the effects of its inhibition are yet to be consistently determined. RIPK3-MLKL-mediated necroptosis also contributed to ischemia-reperfusion injury of steatotic livers.
      • Ni H.M.
      • Chao X.
      • Kaseff J.
      • Deng F.
      • Wang S.
      • Shi Y.H.
      • et al.
      Receptor-Interacting Serine/Threonine-Protein Kinase 3 (RIPK3)-Mixed Lineage Kinase Domain-Like Protein (MLKL)-mediated necroptosis contributes to ischemia-reperfusion injury of steatotic livers.
      Mlkl-/- mice had decreased hepatic neutrophil infiltration and inflammation, while Ripk3-/- or RIPK3 kinase-dead knock-in mice were protected against late ischemia-reperfusion injury.
      Interestingly, RIPK3 deficiency protected MCD diet-fed mice from liver injury, inflammation and fibrosis,
      • Afonso M.B.
      • Rodrigues P.M.
      • Carvalho T.
      • Caridade M.
      • Borralho P.
      • Cortez-Pinto H.
      • et al.
      Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis.
      ,
      • Gautheron J.
      • Vucur M.
      • Reisinger F.
      • Cardenas D.V.
      • Roderburg C.
      • Koppe C.
      • et al.
      A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis.
      but exacerbated liver steatosis and apoptosis, as well as adipose tissue inflammation, insulin resistance and glucose intolerance induced by high-fat diet.
      • Gautheron J.
      • Vucur M.
      • Schneider A.T.
      • Severi I.
      • Roderburg C.
      • Roy S.
      • et al.
      The necroptosis-inducing kinase RIPK3 dampens adipose tissue inflammation and glucose intolerance.
      ,
      • Roychowdhury S.
      • McCullough R.L.
      • Sanz-Garcia C.
      • Saikia P.
      • Alkhouri N.
      • Matloob A.
      • et al.
      Receptor interacting protein 3 protects mice from high-fat diet-induced liver injury.
      While, RIPK3 global knockout mice exhibit no phenotype, the mechanism underlying enhanced apoptosis in Ripk3-/- mice on a high-fat diet is uncertain, and contradictory to that reported earlier in an alcohol model.
      • Roychowdhury S.
      • McMullen M.R.
      • Pisano S.G.
      • Liu X.
      • Nagy L.E.
      Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury.
      Nevertheless, Ripk3-/- mice fed a choline-deficient high-fat diet developed insulin resistance as a result of increased compensatory apoptosis and inflammation in white adipose tissue.
      • Gautheron J.
      • Vucur M.
      • Schneider A.T.
      • Severi I.
      • Roderburg C.
      • Roy S.
      • et al.
      The necroptosis-inducing kinase RIPK3 dampens adipose tissue inflammation and glucose intolerance.
      The protective function of RIPK3 in adipose tissue argues in favour of hepatocyte-specific RIPK3 inhibition in NASH. Moreover, if the increased hepatocellular apoptosis is not directly determined by the absence of RIPK3 in hepatocytes, a non-parenchymal anti-inflammatory role of RIPK3 should be further dissected using conditional knockout models. Other functions of RIPK3 in hepatocarcinogenesis have been recognised,
      • Vucur M.
      • Reisinger F.
      • Gautheron J.
      • Janssen J.
      • Roderburg C.
      • Cardenas D.V.
      • et al.
      RIP3 inhibits inflammatory hepatocarcinogenesis but promotes cholestasis by controlling caspase-8- and JNK-dependent compensatory cell proliferation.
      and its involvement in inflammatory signalling independent of necroptosis and mediation of cell death should not be neglected.
      • Moriwaki K.
      • Chan F.K.
      Necrosis-dependent and independent signaling of the RIP kinases in inflammation.
      ,
      • Newton K.
      RIPK1 and RIPK3: critical regulators of inflammation and cell death.
      RIPK3-deficient natural killer cells showed impaired immune responses to tumours and liver inflammation.
      • Kang Y.J.
      • Bang B.R.
      • Han K.H.
      • Hong L.
      • Shim E.J.
      • Ma J.
      • et al.
      Regulation of NKT cell-mediated immune responses to tumours and liver inflammation by mitochondrial PGAM5-Drp1 signalling.
      Interestingly, the strong induction of RIPK3 and MLKL in experimental models of NASH and in patients with chronic liver disease is required to sensitise cells to necroptosis. Epigenetic silencing of RIPK3, in contrast, has been associated with malignant transformation in hepatocellular carcinoma cells, while reestablishment of RIPK3 expression by promoter demethylation resensitised cells to chemotherapy-induced necroptosis,
      • Koo G.B.
      • Morgan M.J.
      • Lee D.G.
      • Kim W.J.
      • Yoon J.H.
      • Koo J.S.
      • et al.
      Methylation-dependent loss of RIP3 expression in cancer represses programmed necrosis in response to chemotherapeutics.
      again implying that induction of RIPK3 is context-dependent.
      Overall, RIP kinases are pleiotropic modulators of cell death that participate in the pathogenesis of many chronic diseases,
      • He S.
      • Wang X.
      RIP kinases as modulators of inflammation and immunity.
      suggesting that preventing the formation and/or activation of the necrosome might arrest disease progression in NASH. In a recent study, RIPA-56, a highly specific RIPK1 inhibitor, reduced hepatic inflammation and fibrosis in dietary obese mice, while reversing steatosis and dampening body weight gain.
      • Majdi A.
      • Aoudjehane L.
      • Ratziu V.
      • Islam T.
      • Afonso M.B.
      • Conti F.
      • et al.
      Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease.
      Similarly, pharmacological inhibition of MLKL decreased hepatic de novo fat synthesis and chemokine ligand expression in NASH.
      • Saeed W.K.
      • Jun D.W.
      • Jang K.
      • Oh J.H.
      • Chae Y.J.
      • Lee J.S.
      • et al.
      Decrease in fat de novo synthesis and chemokine ligand expression in non-alcoholic fatty liver disease caused by inhibition of mixed lineage kinase domain-like pseudokinase.
      Further studies in human NAFLD and NASH using robust hallmarks of necroptosis, as well as intervention studies in conditional knockouts and in models that better mimic the same degree of inflammation and fibrosis as human disease should provide additional insights on the therapeutic relevance of targeting necroptosis in the context of NAFLD.

      Pyroptosis

      Lipotoxicity, innate immune response, cell death pathways, mitochondrial dysfunction and endoplasmic reticulum stress trigger chronic inflammation in the liver, potentially fuelling the transition from NAFL to NASH.
      • Farrell G.C.
      • van Rooyen D.
      • Gan L.
      • Chitturi S.
      NASH is an inflammatory disorder: pathogenic, prognostic and therapeutic implications.
      • Mridha A.R.
      • Wree A.
      • Robertson A.A.B.
      • Yeh M.M.
      • Johnson C.D.
      • Van Rooyen D.M.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      • Schuster S.
      • Cabrera D.
      • Arrese M.
      • Feldstein A.E.
      Triggering and resolution of inflammation in NASH.
      In this regard, activation of resident Kupffer cells and infiltrating macrophages is a remarkable feature of NASH pathogenesis.
      • Syn W.K.
      • Oo Y.H.
      • Pereira T.A.
      • Karaca G.F.
      • Jung Y.
      • Omenetti A.
      • et al.
      Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease.
      While Kupffer cells are known to release TNF-α,
      • Tosello-Trampont A.C.
      • Landes S.G.
      • Nguyen V.
      • Novobrantseva T.I.
      • Hahn Y.S.
      Kuppfer cells trigger nonalcoholic steatohepatitis development in diet-induced mouse model through tumor necrosis factor-alpha production.
      further feeding a vicious cycle of inflammation and apoptosis leading to fibrosis, it is now apparent that inflammatory caspases, including caspase-1, murine caspase-11, and human caspase-4/5, play important roles as mediators of inflammation.
      • Shi J.
      • Zhao Y.
      • Wang K.
      • Shi X.
      • Wang Y.
      • Huang H.
      • et al.
      Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.
      This implicates pyroptosis in non-alcoholic fatty liver development and progression to NASH.
      Inflammasome activation triggers caspase activity and promotes inflammation and fibrosis in liver disease,
      • Szabo G.
      • Petrasek J.
      Inflammasome activation and function in liver disease.
      while typical activators of inflammasomes such as fatty acids, DAMPs, and uric acid upregulate NLRP3 inflammasome components. Excessive activation of inflammatory caspases has been directly implicated in the pathogenesis of NAFLD in humans and mice, where proinflammatory cytokines released during pyroptosis are key effector molecules.
      • Szabo G.
      • Petrasek J.
      Inflammasome activation and function in liver disease.
      • Henao-Mejia J.
      • Elinav E.
      • Jin C.
      • Hao L.
      • Mehal W.Z.
      • Strowig T.
      • et al.
      Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity.
      • Xu B.
      • Jiang M.
      • Chu Y.
      • Wang W.
      • Chen D.
      • Li X.
      • et al.
      Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice.
      Indeed, activation of IL-1β signalling, downstream of inflammasomes, drives liver inflammation and fibrosis in experimental NASH
      • Mridha A.R.
      • Wree A.
      • Robertson A.A.B.
      • Yeh M.M.
      • Johnson C.D.
      • Van Rooyen D.M.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      and amplifies the response of other cytokines.
      • Tilg H.
      • Moschen A.R.
      • Szabo G.
      Interleukin-1 and inflammasomes in alcoholic liver disease/acute alcoholic hepatitis and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis.
      Another generic substrate for inflammatory caspases, GSDMD, acts as a downstream effector of non-canonical inflammasome activation and plays a specific role in inflammatory caspase-mediated pyroptosis.
      • Broz P.
      • Pelegrin P.
      • Shao F.
      The gasdermins, a protein family executing cell death and inflammation.
      The GSDMDNT domain with intrinsic pyroptosis-inducing activity was positively correlated with the NAFLD activity score and fibrosis in patients with NASH.
      • Xu B.
      • Jiang M.
      • Chu Y.
      • Wang W.
      • Chen D.
      • Li X.
      • et al.
      Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice.
      Further, Gsdmd−/− mice fed an MCD diet showed significantly reduced steatohepatitis, noticeable improvement in liver inflammation, as well as reduced serum alanine aminotransferase (ALT) levels and hepatic triglyceride content. In addition, liver fibrosis was strongly attenuated in Gsdmd−/− mice after MCD feeding.
      • Xu B.
      • Jiang M.
      • Chu Y.
      • Wang W.
      • Chen D.
      • Li X.
      • et al.
      Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice.
      Interestingly, Gsdmd−/− mice were protected from steatosis via downregulation of the lipogenic gene Srebp1c and induction of lipolytic genes, including Pparα and its downstream targets. Importantly, overexpression of the GSDMDNT domain, functionally responsible for pyroptosis, spontaneously induced liver injury even without MCD treatment, indicating that GSDMDNT-induced pyroptosis is a crucial mechanism involved in the pathogenesis of steatohepatitis.
      PAMPs and DAMPs can directly induce pyroptotic cell death in hepatocytes or indirectly cause liver cell injury. Interestingly, compared to global NLRP3 knock-in mice, mice with myeloid-specific Nlrp3 mutations lack detectable pyroptotic hepatocyte cell death and have less severe liver inflammation, HSC activation, and fibrosis.
      • Wree A.
      • Eguchi A.
      • McGeough M.D.
      • Pena C.A.
      • Johnson C.D.
      • Canbay A.
      • et al.
      NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.
      Thus, in addition to immune cells, hepatocyte pyroptosis resulting from intrinsic inflammasome activation exacerbates inflammation and fibrosis in the liver, indicating that both immune cell- and liver-specific NLRP3 inflammasome activation are essential for liver injury.
      • Wree A.
      • Eguchi A.
      • McGeough M.D.
      • Pena C.A.
      • Johnson C.D.
      • Canbay A.
      • et al.
      NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.
      ,
      • Wree A.
      • McGeough M.D.
      • Inzaugarat M.E.
      • Eguchi A.
      • Schuster S.
      • Johnson C.D.
      • et al.
      NLRP3 inflammasome driven liver injury and fibrosis: roles of IL-17 and TNF in mice.
      Hepatocyte-specific NLRP3 mutant animals are nevertheless needed for direct evidence of the crosstalk between hepatocytes and the other cell types in the onset and progression of liver injury in NAFLD and NASH.

      Ferroptosis

      Evidence for a potential role of ferroptosis in NAFLD and NASH is scarce. Nevertheless, secondary products of lipid peroxidation, such as malondialdehyde and 4-hydroxinonenal can be utilised as oxidative stress markers in patients with NASH,
      • Loguercio C.
      • De Girolamo V.
      • de Sio I.
      • Tuccillo C.
      • Ascione A.
      • Baldi F.
      • et al.
      Non-alcoholic fatty liver disease in an area of southern Italy: main clinical, histological, and pathophysiological aspects.
      while Vitamin E, an antioxidant that suppresses lipid peroxidation, reduces serum ALT in patients with NASH.
      • Sanyal A.J.
      • Chalasani N.
      • Kowdley K.V.
      • McCullough A.
      • Diehl A.M.
      • Bass N.M.
      • et al.
      Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis.
      Moreover, iron accumulation from metabolic dysfunction, aggravates NASH, such as in liver siderosis observed in some patients with NASH,
      • Nelson J.E.
      • Wilson L.
      • Brunt E.M.
      • Yeh M.M.
      • Kleiner D.E.
      • Unalp-Arida A.
      • et al.
      Relationship between the pattern of hepatic iron deposition and histological severity in nonalcoholic fatty liver disease.
      and primary hemochromatosis exacerbates NASH,
      • Bonkovsky H.L.
      • Jawaid Q.
      • Tortorelli K.
      • LeClair P.
      • Cobb J.
      • Lambrecht R.W.
      • et al.
      Non-alcoholic steatohepatitis and iron: increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis.
      while iron removal ameliorates liver damage and serum ALT.
      • Valenti L.
      • Moscatiello S.
      • Vanni E.
      • Fracanzani A.L.
      • Bugianesi E.
      • Fargion S.
      • et al.
      Venesection for non-alcoholic fatty liver disease unresponsive to lifestyle counselling--a propensity score-adjusted observational study.
      In a recent study, inhibitors of ferroptosis, trolox and deferiprone, suppressed necrotic cell death, infiltration of inflammatory cells, and inflammatory cytokine expression at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model,
      • Tsurusaki S.
      • Tsuchiya Y.
      • Koumura T.
      • Nakasone M.
      • Sakamoto T.
      • Matsuoka M.
      • et al.
      Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis.
      in which hepatic cell death is an early event in the context of steatosis.
      • Kohn-Gaone J.
      • Dwyer B.J.
      • Grzelak C.A.
      • Miller G.
      • Shackel N.A.
      • Ramm G.A.
      • et al.
      Divergent inflammatory, fibrogenic, and liver progenitor cell dynamics in two common mouse models of chronic liver injury.
      Oxygenated phosphatidylethanolamine, implicated in the ferroptosis pathway, was increased in the liver of CDE-fed mice, but normalized after trolox treatment. Consistently, the hepatic phosphatidylcholine/phosphatidylethanolamine ratio is decreased in patients with NASH.
      • Li Z.
      • Agellon L.B.
      • Allen T.M.
      • Umeda M.
      • Jewell L.
      • Mason A.
      • et al.
      The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis.
      ,
      • Hernandez-Alvarez M.I.
      • Sebastian D.
      • Vives S.
      • Ivanova S.
      • Bartoccioni P.
      • Kakimoto P.
      • et al.
      Deficient endoplasmic reticulum-mitochondrial phosphatidylserine transfer causes liver disease.
      Overall, the potential role of ferroptosis in NAFLD and NASH warrants further exploration in meaningful models of disease and in patients, particularly since no therapeutic options are currently available for NASH.
      The regulation of different pathways of cell death is complicated. Therapeutic targeting of one mode of cell death could enhance or inhibit another.

      Alcohol-associated liver disease and alcoholic steatohepatitis

      Alcohol-associated liver disease (ALD) is a major health problem worldwide and a significant source of liver injury, characterised by a wide spectrum of hepatic lesions from steatosis, steatohepatitis and fibrosis that may progress to cirrhosis and even hepatocellular carcinoma.
      • Bataller R.
      • Gao B.
      Liver fibrosis in alcoholic liver disease.
      Cell injury, inflammation, oxidative stress, regeneration and bacterial translocation are key drivers of alcohol-induced liver injury.
      • Louvet A.
      • Mathurin P.
      Alcoholic liver disease: mechanisms of injury and targeted treatment.
      ALD develops via a complex process involving parenchymal and non-parenchymal cells, as well as recruitment of other cell types to the liver in response to damage and inflammation. Importantly, cross-regulation by the effectors of different cell death pathways most likely influences liver inflammation in ALD. It is possible that apoptosis occurs in early ALD-related steatosis, followed by necroptosis in early ASH and pyroptosis in alcoholic hepatitis, in agreement with neutrophilic inflammation and endotoxemia and septicaemia, the most common cause of death from alcoholic hepatitis.

      Necroptosis

      The involvement of necroptosis has been suggested from studies where RIPK3 expression was induced in human ALD and in mice after ethanol exposure.
      • Roychowdhury S.
      • McMullen M.R.
      • Pisano S.G.
      • Liu X.
      • Nagy L.E.
      Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury.
      ,
      • Wang S.
      • Ni H.M.
      • Dorko K.
      • Kumer S.C.
      • Schmitt T.M.
      • Nawabi A.
      • et al.
      Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.
      RIPK3-mediated necroptosis and neutrophil-mediated liver inflammatory responses were also highly correlated with poor prognosis in patients with end-stage alcoholic cirrhosis.
      • Zhang Z.
      • Xie G.
      • Liang L.
      • Liu H.
      • Pan J.
      • Cheng H.
      • et al.
      RIPK3-mediated necroptosis and neutrophil infiltration are associated with poor prognosis in patients with alcoholic cirrhosis.
      RIPK3 deficiency, in turn, effectively protected against ethanol-induced serum liver enzyme abnormalities, steatosis and inflammation.
      • Roychowdhury S.
      • McMullen M.R.
      • Pisano S.G.
      • Liu X.
      • Nagy L.E.
      Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury.
      ,
      • Wang S.
      • Ni H.M.
      • Dorko K.
      • Kumer S.C.
      • Schmitt T.M.
      • Nawabi A.
      • et al.
      Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.
      Nevertheless, inhibition of RIP1 kinase activity blunted Gao-binge alcohol-induced hepatic inflammation but did not protect against chronic ethanol feeding-induced steatosis and liver injury, suggesting that alcohol-induced RIPK3-mediated necroptosis is independent of RIPK1.
      • Wang S.
      • Ni H.M.
      • Dorko K.
      • Kumer S.C.
      • Schmitt T.M.
      • Nawabi A.
      • et al.
      Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury.
      Other studies reported that ethanol-induced decreases in Nrf2 expression were abrogated by curcumin or gallic acid treatment, by suppressing RIPK3 and RIPK1 expression as well as DAMP release.
      • Lu C.
      • Xu W.
      • Zhang F.
      • Shao J.
      • Zheng S.
      Nrf2 knockdown disrupts the protective effect of curcumin on alcohol-induced hepatocyte necroptosis.
      ,
      • Zhou Y.
      • Jin H.
      • Wu Y.
      • Chen L.
      • Bao X.
      • Lu C.
      Gallic acid protects against ethanol-induced hepatocyte necroptosis via an NRF2-dependent mechanism.
      Overall, RIPK1 involvement remains to be consistently demonstrated in ethanol-induced hepatic injury, RIPK3 depletion should be attempted in other relevant cell types, and MLKL targeting warrants further exploration. While it appears clear that ethanol-induced necroptosis involves RIPK3, it is not yet known if MLKL-independent pathways are involved as reported in models of autoimmune arthritis.
      • Lawlor K.E.
      • Khan N.
      • Mildenhall A.
      • Gerlic M.
      • Croker B.A.
      • D'Cruz A.A.
      • et al.
      RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL.
      Further, increased infiltration of inflammatory cells in the liver after alcohol exposure requires the inclusion of inflammatory cell-specific knockout mice in future studies to clarify the role of necroptosis in alcohol-induced liver pathogenesis.

      Pyroptosis

      Excessive and uncontrolled activation of inflammasomes has a damaging effect on the host, inducing uncontrolled inflammatory responses and the consequent cell death. Recent evidence shows that pyroptosis plays a prominent role in ALD pathogenesis. The NLRP3 inflammasome pathway is activated in hepatocytes in response to endotoxin challenge, a condition facilitated by alcohol intake.
      • Boaru S.G.
      • Borkham-Kamphorst E.
      • Tihaa L.
      • Haas U.
      • Weiskirchen R.
      Expression analysis of inflammasomes in experimental models of inflammatory and fibrotic liver disease.
      Consistently, NLRP3 deficiency ameliorated alcohol-driven liver steatosis and injury,
      • Petrasek J.
      • Iracheta-Vellve A.
      • Saha B.
      • Satishchandran A.
      • Kodys K.
      • Fitzgerald K.A.
      • et al.
      Metabolic danger signals, uric acid and ATP, mediate inflammatory cross-talk between hepatocytes and immune cells in alcoholic liver disease.
      while caspase-4/11 was upregulated in liver tissue from patients with alcoholic hepatitis and in mice, respectively.
      • Khanova E.
      • Wu R.
      • Wang W.
      • Yan R.
      • Chen Y.
      • French S.W.
      • et al.
      Pyroptosis by caspase11/4-gasdermin-D pathway in alcoholic hepatitis in mice and patients.
      Pyroptosis triggered by gut-derived PAMPS and by metabolic-derived DAMPs (ATP and uric acid) resulted in release of inflammasome-dependent cytokines from immune cells in patients with ALD and in cells exposed to ethanol.
      • Petrasek J.
      • Iracheta-Vellve A.
      • Saha B.
      • Satishchandran A.
      • Kodys K.
      • Fitzgerald K.A.
      • et al.
      Metabolic danger signals, uric acid and ATP, mediate inflammatory cross-talk between hepatocytes and immune cells in alcoholic liver disease.
      ,
      • Shulga N.
      • Pastorino J.G.
      Hexokinase II binding to mitochondria is necessary for Kupffer cell activation and is potentiated by ethanol exposure.
      Caspase-1 was also activated in hepatocytes of patients with alcoholic hepatitis.
      • Peng Y.
      • French B.A.
      • Tillman B.
      • Morgan T.R.
      • French S.W.
      The inflammasome in alcoholic hepatitis: its relationship with Mallory-Denk body formation.
      Interestingly, a recent study demonstrated that alcohol elicits caspase-1-mediated pyroptosis through overexpression of thioredoxin-interacting protein, a member of the α-arrestin family; this event is reversed by miR-148a.
      • Heo M.J.
      • Kim T.H.
      • You J.S.
      • Blaya D.
      • Sancho-Bru P.
      • Kim S.G.
      Alcohol dysregulates miR-148a in hepatocytes through FoxO1, facilitating pyroptosis via TXNIP overexpression.
      MiR-148a levels, in turn, are deregulated in the liver of patients with alcoholic hepatitis or ethanol-fed animals. Consistently, hepatocyte-specific delivery of miR-148a alleviates alcoholic liver injury. These data together with the fact that serum ALT activities remained elevated after macrophage depletion support the direct effect of alcohol on NLRP3 inflammasome activation in hepatocytes. Pyroptosis is thus able to stimulate and effectively sustain the inflammatory cycle in ALD, although the specific contribution of the NLRP3 inflammasome in immune cells and hepatic stellate cells remains debatable.

      Ferroptosis

      Iron overload and increased oxidative stress have been well documented in ALD.
      • Nagy L.E.
      • Ding W.X.
      • Cresci G.
      • Saikia P.
      • Shah V.H.
      Linking pathogenic mechanisms of alcoholic liver disease with clinical phenotypes.
      Recent findings shed light on the mechanisms underlying alcohol-induced liver damage by showing that adipose-specific lipin-1 overexpression accelerated iron accumulation, caused lipid peroxidation, reduced GSH and GAPDH, and promoted ferroptotic liver damage in mice after ethanol administration.
      • Zhou Z.
      • Ye T.J.
      • Bonavita G.
      • Daniels M.
      • Kainrad N.
      • Jogasuria A.
      • et al.
      Adipose-specific lipin-1 overexpression renders hepatic ferroptosis and exacerbates alcoholic steatohepatitis in mice.
      Lipin-1 is a Mg2+-dependent phosphatidic acid phosphohydrolase involved in the generation of diacylglycerol during synthesis of phospholipids and triglycerides. However, it remains to be elucidated how hepatic ferroptotic signalling is involved in this pathogenic process.

      Other metabolic liver disorders

      Haemochromatosis

      Studies on cell death in other metabolic liver disorders, including iron-overload diseases, and genetic disorders such as Niemann-Pick disease or Gaucher's disease are scarce. Iron is implicated in the pathogenesis of a number of human liver diseases. Hereditary haemochromatosis is the classical example of a liver disease caused by iron, but iron is an important contributor to the progression of other forms of chronic liver disease, including NAFLD. Iron-induced liver damage is likely potentiated by coexisting inflammation, which culminates with hepatocyte cell death and drives fibrogenesis.
      • Bloomer S.A.
      • Brown K.E.
      Iron-induced liver injury: a critical reappraisal.
      Hereditary haemochromatosis is caused by mutations in genes whose protein products limit iron absorption. Consequently, excess iron generates ROS thereby inducing cell death and global oxidative damage, ultimately leading to severe chronic complications, including cirrhosis. Nevertheless, the precise roles of iron and iron metabolism in cell death are largely unknown. A recent study investigated the role of iron homeostasis in SLC7A11-mediated ferroptosis and found that iron overload triggers ferroptosis both in vitro and in vivo.
      • Wang H.
      • An P.
      • Xie E.
      • Wu Q.
      • Fang X.
      • Gao H.
      • et al.
      Characterization of ferroptosis in murine models of hemochromatosis.
      Indeed, Slc7a11 knockout was not sufficient to induce ferroptosis under basal conditions but facilitated iron overload-induced ferroptosis due to impaired cystine uptake and increased ROS production. Ferroptosis is thus a potential therapeutic target for treating iron overload-associated diseases, including haemochromatosis. Other lytic forms of cell death need to be explored in haemochromatosis and other metal-related liver disorders.

      Niemann-Pick disease

      Niemann-Pick disease, type C1 (NPC1) is an inborn error of metabolism that results in endolysosomal accumulation of unesterified cholesterol. Clinically, NPC1 manifests as cholestatic liver disease in the newborn or as a progressive neurodegenerative condition characterised by cerebellar ataxia and cognitive decline. Neuroinflammation and necroptosis contribute to the pathological cascade. Pharmacological and genetic inhibition of RIPK1's kinase activity increased lifespan in both Npc1−/− mice treated with RIPK1 inhibitors, and Npc1−/−/Ripk1 kinase dead double-mutant mice.
      • Cougnoux A.
      • Cluzeau C.
      • Mitra S.
      • Li R.
      • Williams I.
      • Burkert K.
      • et al.
      Necroptosis in Niemann-Pick disease, type C1: a potential therapeutic target.
      Nevertheless, the increase in lifespan was modest, suggesting that the therapeutic potential of RIPK1 inhibition, as a monotherapy, is limited. Conversely, no increased survival was noted in Npc1−/−/Ripk3−/− mice compared to Npc1−/− mice. These data suggest that although necroptosis is occurring in NPC1, the effects of RIPK1 inhibition may be related to its RIPK3-independent role in neuroinflammation and cytokine production. Nevertheless, corroborating the role of alternative RCD sub-routines, cellular death of NPC1 fibroblasts was not mitigated by treatment with caspase inhibitors
      • Cougnoux A.
      • Cluzeau C.
      • Mitra S.
      • Li R.
      • Williams I.
      • Burkert K.
      • et al.
      Necroptosis in Niemann-Pick disease, type C1: a potential therapeutic target.
      or following neuronal overexpression of Bcl-2.
      • Erickson R.P.
      • Bernard O.
      Studies on neuronal death in the mouse model of Niemann-Pick C disease.
      More importantly, necroptosis inhibition and combination therapy with 2-hydroxypropyl-β-cyclodextrin has recently been suggested for NPC1.
      • Cougnoux A.
      • Clifford S.
      • Salman A.
      • Ng S.L.
      • Bertin J.
      • Porter F.D.
      Necroptosis inhibition as a therapy for Niemann-Pick disease, type C1: inhibition of RIP kinases and combination therapy with 2-hydroxypropyl-beta-cyclodextrin.

      Gaucher's disease

      Necroptosis appears to have a role in the pathological cascade of other lysosomal storage diseases. Gaucher’s disease is an inherited metabolic disorder caused by mutations in the glucocerebrosidase gene (GBA), and the most common lysosomal storage disease. Neuronal cell death in mice with Gaucher’s disease is caspase-independent and non-apoptotic. In striking contrast, RIPK3-mediated necroptosis has been implicated in the pathology of Gaucher's disease.
      • Vitner E.B.
      • Salomon R.
      • Farfel-Becker T.
      • Meshcheriakova A.
      • Ali M.
      • Klein A.D.
      • et al.
      RIPK3 as a potential therapeutic target for Gaucher's disease.
      In fact, levels of Ripk1 and Ripk3 were markedly upregulated in Gaucher’s disease brains, both in mice and in patients, but were unaltered in brains obtained from mouse models of NPC1. This indicates that although brain inflammation and microglial activation are shared features of many lysosomal storage diseases, pathways of neuroinflammation are disease specific. Moreover, RIPK3 deficiency improved the clinical course in mice with Gaucher's disease, with improved survival and motor coordination, and beneficial effects on cerebral and hepatic injury. Ablation of Ripk3 resulted in fewer CD68-positive Kupffer cells and decreased ALT activity, suggesting that hepatocyte injury was also attenuated. Development of necroptosis inhibitors with improved pharmacokinetics may pave the way for alternative therapeutic approaches for Gaucher's disease, for which innovative treatment is urgently required. The specific involvement of other RCD forms is yet to be determined.
      Functional characterisation of lytic cell death in appropriate models of metabolic liver disease and in patients might result in novel therapeutic strategies that target regulated hepatocyte cell death to prevent disease progression.

      Controversies and unsolved issues

      Perspective of cell death in liver diseases

      Cell death has been described as a clear and present danger for hepatocytes as they are directly exposed to portal blood from the intestines, participate in the xenobiotic metabolism of drugs and alcohol, play a central role in lipid, fatty acid, and bile acid metabolism, have contact with prevalent hepatotropic viruses, and reside within a milieu of innate immune-responding cells.
      • Malhi H.
      • Guicciardi M.E.
      • Gores G.J.
      Hepatocyte death: a clear and present danger.
      Yet elucidating the precise role for various modes of cell death in liver diseases has proven difficult, and unpredictable. For example, apoptosis as a driving force in liver injury and carcinogenesis has only recently been established despite intense study over the last 2 decades.
      • Krishna-Subramanian S.
      • Singer S.
      • Armaka M.
      • Banales J.M.
      • Holzer K.
      • Schirmacher P.
      • et al.
      RIPK1 and death receptor signaling drive biliary damage and early liver tumorigenesis in mice with chronic hepatobiliary injury.
      It will also take time and detailed analysis of relevant models to identify the potency and hierarchal relationship between apoptosis, necroptosis, pyroptosis and ferroptosis in liver diseases. Additionally, therapeutic perturbations targeting one mode of cell death may potentiate or even inhibit additional modes of liver injury, further complicating this analysis. For example, caspase inhibitors employed to prevent apoptosis, may promote necroptosis by preventing caspase-8 proteolytic degradation of RIPK1, or inhibit pyroptosis by blocking caspase-1 or -8. In this section, we will acknowledge these controversies, providing nuanced insight and a critical appraisal of the information described earlier.

      Unresolved issues regarding necroptosis in liver diseases

      The role of necroptosis in liver diseases remains to be firmly established. This uncertainty relates to current tools, models, and evolving information that permits a reinterpretation of prior studies. First, there is vigorous controversy regarding the expression of RIPK3 by hepatocytes, but under basal conditions expression appears to be quite limited.
      • Dara L.
      • Liu Z.X.
      • Kaplowitz N.
      Questions and controversies: the role of necroptosis in liver disease.
      ,
      • Dara L.
      The receptor interacting protein kinases in the liver.
      Whether they can be induced in various liver diseases remains unclear, as the antibodies employed for immunoblot analysis and immunohistochemistry are fraught with non-specificity. We are not aware of any detailed proteomic studies which have been performed to address these questions, although the technology to examine phosphoproteins by this approach is widely available. Moreover, activating phosphorylation of RIPK3 and MLKL have been identified in the absence of cell death, questioning the use of these phosphoproteins as biomarkers for necroptosis.
      • Dara L.
      • Liu Z.X.
      • Kaplowitz N.
      Questions and controversies: the role of necroptosis in liver disease.
      Second, RIPK3 deficiency does not phenocopy MLKL deficiency, and hence studies with RIPK3 knockout or kinase dead knock-in mice, are not sufficient to invoke necroptosis as a cause of cell death, as RIPK3 has diverse functions.
      • Moriwaki K.
      • Chan F.K.
      RIP3: a molecular switch for necrosis and inflammation.
      Moreover, inhibition of RIPK3 often provokes apoptosis, further confusing attempts to examine necroptosis in disease models.
      • Mandal P.
      • Berger S.B.
      • Pillay S.
      • Moriwaki K.
      • Huang C.
      • Guo H.
      • et al.
      RIP3 induces apoptosis independent of pronecrotic kinase activity.
      The multifunctional aspect of RIPK3 biology in different cell types certainly needs to be considered when germline- vs. cell type-specific knockout or kinase dead knock-in paradigms are employed. Third, caspase-8 limits necroptosis by proteolytically cleaving RIPK1,
      • Newton K.
      • Wickliffe K.E.
      • Dugger D.L.
      • Maltzman A.
      • Roose-Girma M.
      • Dohse M.
      • et al.
      Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis.
      and the genetic absence of caspase-8 in hepatocytes does not trigger wide-spread necroptosis.
      • Krishna-Subramanian S.
      • Singer S.
      • Armaka M.
      • Banales J.M.
      • Holzer K.
      • Schirmacher P.
      • et al.
      RIPK1 and death receptor signaling drive biliary damage and early liver tumorigenesis in mice with chronic hepatobiliary injury.
      Finally, genetic studies employing Ripk3 knockout mice suggest that cholangiocyte but not hepatocyte injury is ameliorated, indicating that necroptosis mediates cholangiocyte but not hepatocyte injury.
      • Krishna-Subramanian S.
      • Singer S.
      • Armaka M.
      • Banales J.M.
      • Holzer K.
      • Schirmacher P.
      • et al.
      RIPK1 and death receptor signaling drive biliary damage and early liver tumorigenesis in mice with chronic hepatobiliary injury.
      These observations will need to be reconciled with those implicating necroptosis in liver disease (see future directions below).

      Unresolved issues regarding pyroptosis in liver diseases

      Pyroptosis requires caspase-dependent cleavage of GSDMD to plasma membrane pore-forming fragments.
      • Kayagaki N.
      • Dixit V.M.
      Rescue from a fiery death: a therapeutic endeavor.
      Cleavage of GSDMD occurs via a canonical inflammasome caspase-1 dependent mechanism, a non-canonical inflammasome pathway involving caspases -4, -5 or -11,
      • Kayagaki N.
      • Dixit V.M.
      Rescue from a fiery death: a therapeutic endeavor.
      or in an inflammasome-independent caspase-8 mediated pathway.
      • Orning P.
      • Lien E.
      • Fitzgerald K.A.
      Gasdermins and their role in immunity and inflammation.
      Although all cell types appear to be inflammasome competent, the best examples are in cells of the innate immune system, especially those interacting with intracellular organisms. Cell type-specific GSDMD will be required to examine this form of cell death in hepatocytes. Interestingly, in shock models of liver injury, mice with a germline deficiency in Gsdmd display increased caspase-8 activation and apoptosis in the liver.
      • Yang C.
      • Sun P.
      • Deng M.
      • Loughran P.
      • Li W.
      • Yi Z.
      • et al.
      Gasdermin D protects against noninfectious liver injury by regulating apoptosis and necroptosis.
      This is another cautionary example where inhibiting one mode of cell death enhances a different mechanism of liver injury.

      Unresolved issues regarding ferroptosis in liver diseases

      Oxidative stress has long-been implicated in liver injury. Ferroptosis which is regulated by the availability of cytosolic free iron, cystine transport into the cell, GPX4 activity, and GSH levels, leads to cell death by lipid peroxidation. A specific linchpin molecular executioner of ferroptosis has not been described. Hence genetic deletion of a final common pathway mediator is not yet feasible. In fact, propagation of lipid peroxidation is non-enzymatic and may prove difficult to target. In this regard, the role of ferroptosis in liver disease will be largely circumstantial, and we note that, to date, antioxidant therapy has not been impactful in human liver diseases. Careful work will be necessary to dissect the ultimate role of ferroptosis in liver diseases.

      Future directions and therapeutic implications

      The contribution of different modes of cell death to various liver diseases will require carefully defined preclinical studies that address causality. These studies will need to focus on the terminal executioners of cell death, as upstream pathway mediators have multiple functions. For example, RIP kinases have diverse functions in addition to promoting necroptosis.
      • He S.
      • Wang X.
      RIP kinases as modulators of inflammation and immunity.
      Such studies need to focus initially on hepatocyte cell death, employing hepatocyte-conditional knockout models of MLKL, GSDMD, and Bax plus Bak, to discern the interrelationships between necroptosis, pyroptosis and apoptosis. These studies will require development of genetic mouse models and hence will take time. Also, conditional knockouts in other cell types (e.g., cholangiocytes and immune cells, such as macrophages) will ultimately be necessary to complete the puzzle of cell death's role in liver injury. Finally, we note that cell death per se may be a biomarker for stressed, proinflammatory hepatocytes as recently suggested in NASH,
      • Ibrahim S.H.
      • Hirsova P.
      • Gores G.J.
      Non-alcoholic steatohepatitis pathogenesis: sublethal hepatocyte injury as a driver of liver inflammation.
      and therefore, precluding cell death may not have the major impact on liver injury that we anticipate.
      These various modes of cell death also represent unique therapeutic avenues for the treatment of cell death in human liver diseases. However, inhibiting one mode of cell death may potentiate other modes of cell death as noted earlier, negating a therapeutic benefit. It was recently reported that caspase inhibition with emricasan treatment did not improve clinical outcomes nor liver histology in patients with NASH-related fibrosis, resulting in more liver fibrosis and hepatocyte ballooning.
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      • et al.
      Randomized placebo-controlled trial of Emricasan in Non-alcoholic Steatohepatitis (NASH) cirrhosis with severe portal hypertension.
      ,
      • Harrison S.A.
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      • Younes Z.H.
      • Freilich B.
      • et al.
      A randomized, placebo-controlled trial of emricasan in patients with NASH and F1-F3 fibrosis.
      Caspase inhibition may have directed cells to alternative, more inflammatory mechanisms of cell death, such as necroptosis. Indeed, emricasan has previously been shown to shift cell death from apoptosis towards necroptosis in acute myeloid leukaemia cells treated with birinapant, a SMAC (second mitochondrial-derived activator of caspases) mimetic, which enhances cancer cell apoptosis.
      • Brumatti G.
      • Ma C.
      • Lalaoui N.
      • Nguyen N.Y.
      • Navarro M.
      • Tanzer M.C.
      • et al.
      The caspase-8 inhibitor emricasan combines with the SMAC mimetic birinapant to induce necroptosis and treat acute myeloid leukemia.
      MLKL inhibitors are being developed such as necrosulfonamide, which binds to the N-terminal 4 helical bundle of MLKL to prevent necroptosis.
      • Bansal N.
      • Sciabola S.
      • Bhisetti G.
      Understanding allosteric interactions in hMLKL protein that modulate necroptosis and its inhibition.
      ,
      • Molnar T.
      • Mazlo A.
      • Tslaf V.
      • Szollosi A.G.
      • Emri G.
      • Koncz G.
      Current translational potential and underlying molecular mechanisms of necroptosis.
      Indeed, a variety of targets and inhibitors are being investigated to block necroptosis, and we refer the reader to this literature.
      • Molnar T.
      • Mazlo A.
      • Tslaf V.
      • Szollosi A.G.
      • Emri G.
      • Koncz G.
      Current translational potential and underlying molecular mechanisms of necroptosis.
      Interestingly, necrosulfonamide also binds GSDMD and blocks pyroptosis.
      • Rathkey J.K.
      • Zhao J.
      • Liu Z.
      • Chen Y.
      • Yang J.
      • Kondolf H.C.
      • et al.
      Chemical disruption of the pyroptotic pore-forming protein gasdermin D inhibits inflammatory cell death and sepsis.
      As these compounds and others are further developed as pharmacologic agents, we look forward to future human clinical trials with anticipation.
      In summary, we now recognise several different forms of cell death, executed by different pathways and mediators. The mechanisms are being unravelled and their contribution to human liver diseases is being carefully examined. These insights reveal further complexity in liver injury, but also provide new and exciting therapeutic opportunities.

      Abbreviations

      ACSL4, acyl-CoA synthetase long chain 4; AIM2, absent in melanoma 2; ALD, alcohol-associated liver disease; ALT, alanine aminotransferase; CARD, caspase recruitment domain; CDE, choline-deficient, ethionine-supplemented; cIAPs, cellular inhibitors of apoptosis; DAMP, damage-associated molecular patterns; DMT1, divalent metal transporter 1; DR, death receptors; dsRNA, double-stranded RNA; ESCRT, endosomal sorting complex required for transport; FADD, FAS-associated protein with death domain; FSP1, ferroptosis suppressor protein 1; GPX4, glutathione peroxidase 4; GSDMD/E, gasdermin D/E; GSH, glutathione; GSSG, glutathione disulphide; HMGB1, high mobility group box 1; IFN, interferon; IFNR, interferon-receptor; IL-, interleukin-; LOXs, lipoxygenases; LPS, lipopolysaccharide; LRR, leucine-rich-repeat; LUBAC, linear ubiquitin chain assembly complex; MCD, methionine- and choline-deficient; MLKL, mixed lineage kinase domain like; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NPC1, Niemann-Pick disease, type C1; PKR, protein kinase R; PYD, pyrin domain; RCD, regulated cell death; RIPK, receptor interacting protein kinase; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SLC, solute carrier; TFRC, transferrin receptor; TAB, transforming growth factor beta-binding protein; TAK, transforming growth factor beta-activated kinase 1; TLR, toll-like receptor; TNF, tumour necrosis factor; TNFR1, TNF receptor 1; TRADD, TNF receptor type 1-associated death domain; TRAIL, TNF-related apoptosis-inducing ligand receptor; TRAF2, TNF receptor-associated factor 2; ZBP1, Z-DNA binding protein 1.

      Financial support

      JG is supported by grants from the Mairie de Paris (Emergences), the Société Francophone du Diabète (SFD), the Institute of Cardiometabolism and Nutrition (ICAN), and the Fondation pour la Recherche Médicale (FRM grant number ARF20170938613 ). GJG is supported by NIH grants DK 124182 and the Digestive Disease Research Center for Cell Signaling in Gastroenterology P30DK084567 , the Chris M Carlos and Catharine Nicole Jockisch Carlos Foundation for PSC, and the Mayo Clinic . CMPR is supported by the Fundação para a Ciência e a Tecnologia (FCT) and European Structural & Investment Funds through the COMPETE Programme (grants UID/DTP/04138/2019 , PTDC/MED-FAR/29097/2017 and SAICTPAC/0019/2015 - LISBOA-01-0145-FEDER-016405 ), and the EU H2020 Marie Sklodowska-Curie Project Foie Gras (grant 722619).

      Authors' contributions

      JG conceived the project, wrote the original draft, reviewed and edited the manuscript; GJG conceived the project, wrote the original draft, reviewed and edited the manuscript; CMPR conceived and administered the project, wrote the original draft, reviewed and edited the manuscript.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgments

      The authors would like to thank Yves Chrétien for expert artwork.

      Supplementary data

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